Process for the measurement of the potency of glatiramer acetate

ABSTRACT

The subject invention provides a process for measuring the relative potency of a test batch of glatiramer acetate. In addition, the subject invention provides a process for preparing a batch of glatiramer acetate as acceptable for pharmaceutical use.

This application claims the benefit of U.S. Provisional Application No.60/338,767, filed Dec. 4, 2001, the contents of which are herebyincorporated by reference.

Throughout this application, various references are cited, usingshortened citations within parentheses. Full citations for thesereferences can be found at the end of the specification, immediatelypreceding the claims. These publications, in their entireties, arehereby incorporated by reference into the application to more fullydescribe the state of the art to which the invention pertains.

FIELD OF THE INVENTION

The present invention relates to methods of standardizing themeasurement of the potency of glatiramer acetate based on specificrecognition of the glatiramer acetate by T cells.

BACKGROUND

It is desirable to standarize the measurement of the potency ofpharmaceutical compositions as there is an optimum potency and qualityof active component that is effective in treating the disease for whichit is administered.

Glatiramer acetate (GA, also known as Copolymer-1 (Physician's DeskReference), Copolymer 1, Cop-1 or COPAXONE®), is an approved drug forthe treatment of multiple sclerosis (MS). Glatiramer acetate consists ofthe acetate salts of synthetic polypeptides, containing four naturallyoccurring amino acids (Physician's Desk Reference): L-glutamic acid,L-alanine, L-tyrosine, nd L-lysine (Physician's Desk Reference) with anaverage molar fraction of L-glutamic acid: 0.129-0.153; L-alanine:0.392-0.462; L-tyrosine: 0.086-0.100; L-lysine: 0.300-0.374,respectively. The average molecular weight of glatiramer acetate is4,700-11,000 daltons (Physician's Desk Reference). Chemically,glatiramer acetate is designated L-glutamic acid polymer with L-alanine,L-lysine and L-tyrosine, acetate (salt) (Physician's Desk Reference).Its structural formula is:

(Glu,Ala,Lys,Tyr)_(x).χCH₃COOH

C

_(H)

_(NO) ₄.C₃H₇NO₂.C₆H₁₄N₂O₂.C₉H₁₁NO₃)_(x).χC₂H₄O₂

CAS-147245-92-9

(Physician's Desk Reference). Glatiramer acetate is also written as:poly[L-Glu¹³⁻¹⁵, L-Ala³⁹⁻⁴⁶, L-Tyr^(8,9,10), L-Lys³⁰⁻³⁷].nCH₃COOH.

Glatiramer acetate was shown to suppress experimental autoimmuneencephalomyelitis (EAE)—an experimental model for multiple sclerosis(MS) in various animal species (Lando et al., 1979; Aharoni, 1993).Studies of murine EAE suggested that the protection against EAE ismediated by T cell activity (Aharoni, 1993). This protection from activeinduction of EAE by mouse spinal cord homogenate, in which severalauto-antigens are involved, could be adoptively transferred to normalrecipients by injection of glatiramer acetate-specific T suppressorcells (Aharoni, 1993). In phase III clinical trials, daily subcutaneousinjections of glatiramer acetate were found to slow progression ofdisability and reduce the relapse rate in exacerbating-remittingmultiple sclerosis (Johnson, 1987). Processes of manufacturingglatiramer acetate are described in U.S. Pat. Nos. 3,849,550 and5,800,808 and PCT International Publication No. WO 00/05250.

It is commonly accepted that a high level of antigen specificity is afeature of T cell activation. The T cells of the immune system recognizeimmunogenic peptides complexed to the major histocompatibility complex(MHC) class II or I molecules, expressed on antigen presenting cells(APCs). The specificity of antigen recognition by T cells is defined byseveral parameters: 1) affinity of the T cell receptor to the MHCpeptide complex; 2) primary sequence of the antigenic peptide; and 3)synergistic effects of certain amino acid combinations within theantigenic peptide. Based on current knowledge on the mechanism of actionof glatiramer acetate, it is believed that the biological activity ofglatiramer acetate in MS is mediated by immunomodulation of T cellactivity.

SUMMARY OF THE INVENTION

The subject invention provides a process for measuring the potency of atest batch of glatiramer acetate relative to the known potency of areference batch of glatiramer acetate which comprises

-   -   a. immunizing female (SJLXBALB/C)F1 mice between 8 and 12 weeks        of age with a predetermined amount of glatiramer acetate from        the reference batch;    -   b. preparing a primary culture of lymph node cells from the mice        of step (a) 9-11 days after immunization;    -   c. separately incubating at least five reference samples, each        of which contains a predetermined number of cells from the        primary culture of step (b) and a predetermined amount of        glatiramer acetate between 1 μg/ml and 25 μg/ml from a reference        batch;    -   d. incubating at least two samples, each of which contains a        predetermined number of cells from the primary culture of        step (b) and a predetermined amount of glatiramer acetate from        the test batch;    -   e. determining for each sample in steps (c) and (d), the amount        of interleukin-2 secreted by the cells in each sample after        18-21 hours of incubation of such sample;    -   f. correlating the amounts of interleukin-2 secreted by the        samples incubated with the test batch of glatiramer acetate with        the amounts of interleukin-2 secreted by the samples incubated        with the reference batch of glatiramer acetate so as to        determine the potency of the test batch of glatiramer acetate        relative to the reference batch of glatiramer acetate,    -   wherein in each sample in steps (c) and (d), the predetermined        number of cells is substantially identical, and wherein for each        sample containing a predetermined amount of glatiramer acetate        from the test batch there is a corresponding reference sample        containing a substantially identical predetermined amount of        glatiramer acetate from the reference batch.

The subject invention also provides a process for measuring the potencyof a test batch of glatiramer acetate relative to the known potency of areference batch of glatiramer acetate which comprises

-   -   a. immunizing a test mammal with a predetermined amount of        glatiramer acetate from the reference batch;    -   b. preparing a primary culture of cells from the test mammal of        step (a) at a predetermined time after immunization;    -   c. separately incubating at least two reference samples, each of        which contains a predetermined number of cells from the primary        culture of step (b) and a predetermined amount of glatiramer        acetate from a reference batch;    -   d. incubating at least two samples, each of which contains a        predetermined number of cells from the primary culture of        step (b) and a predetermined amount of glatiramer acetate from        the test batch;    -   e. determining for each sample in steps (c) and (d), the amount        of a cytokine secreted by the cells in each sample after a        predetermined time period of incubation of such sample;    -   f. correlating the amounts of the cytokine secreted by the        samples incubated with the test batch of glatiramer acetate with        the amounts of the cytokine secreted by the samples incubated        with the reference batch of glatiramer acetate so as to        determine the potency of the test batch of glatiramer acetate        relative to the reference batch of glatiramer acetate,    -   wherein in each sample in steps (c) and (d), the predetermined        number of cells is substantially identical, and wherein for each        immunization sample containing a predetermined amount of        glatiramer acetate from the test batch there is a corresponding        reference sample containing a substantially identical        predetermined amount of glatiramer acetate from the reference        batch.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Immunization with GA RS (Reference Standard). Primary culture ofLN cells. IL-2 Detection by ELISA.

FIG. 2: Induction of GA-specific T cells. Primary cultures of LN cellsderived from mice immunized with 250 μg GA RS+CFA or with CFA alone werecultured in the presence of increasing concentrations of GA RS.Following overnight incubation at 37° C. the culture media werecollected and assayed for IL-2 by ELISA.

FIG. 3: Diagram of hemacytometer.

FIG. 4: Optimization of the immunization protocol—culture source and GAReference Standard (RS) dose effect. Primary cultures of Lymph Node (LN)and spleen cells were derived from mice immunized with 10 or 250 μg GARS+complete Freund's adjuvant (CFA). The cells were cultured in thepresence of increasing concentrations of GA RS. Following overnightincubation at 37° C., the culture media were collected and assayed forIL-2 by enzyme-linked immunoabsorbent assay (ELISA).

FIG. 5: Optimization of the immunization protocol—adjuvant and doseeffect. Primary cultures of LN cells derived from mice immunized with250 μg GA RS+CFA or with 10 mg GA RS+incomplete Freund's adjuvant (ICFA)were cultured in the presence of increasing concentrations of GA RS.Following overnight incubation at 37° C., the culture media werecollected and assayed for interleukin-2 (IL-2) by ELISA.

FIG. 6: Effect of the immunization period. Mice were immunized with 250μg GA RS in CFA and LN were removed after 9, 10 and 11 days. Theresponse of the LN cells from different groups to various concentrationsof GA RS was tested in vitro by measuring IL-2 accretion by ELISA.

FIG. 7: Effect of the culture media on GA-specific T cell response.Primary cultures of LN cells were cultured with different mediacontaining either 1% normal mouse serum (NMS), 1% fetal bovine serum(FBS) or defined cell culture media (DCCM1) The cells were incubatedwith increasing concentrations of GA RS for 21 hours at 37° C.Subsequently, the culture media were collected and assayed for IL-2 byELISA.

FIG. 8: Kinetics of IL-2 secretion in response to GA RS. A primaryculture of LN cells was prepared from mice immunized with 250 μg GARS+CFA. The cells were incubated with 0, 0.5, 2.5 and 5 μg/ml GA RS at37° C. for the indicated intervals. At each time point, an aliquot of5×10⁶ cells was centrifuged and the supernatant was kept at −20° C. Allsamples were assayed simultaneously for IL-2 by ELISA.

FIG. 9: Stability of IL-2 in culture media. A primary culture of LNcells was prepared from mice immunized with 250 μg GA RS+CFA. The cellswere incubated with various concentrations of GA RS at 37°. Afterovernight incubation, the supernatants were collected and divided intotwo aliquots. One aliquot was assayed immediately by ELISA, and thesecond was kept for 7 days at −20° C. prior to being assayed.

FIG. 10: Stability of GA RS solution at −20° C. GA RS solution of 1mg/ml was prepared, divided into aliquots and kept at −20° C. Thedose-response of the GA-specific cells to GA RS solution was tested attime zero (Date 1) and after 5 months at −20° C.

FIG. 11: Effect of the average molecular weight (MW) on the GARS-specific T-cell response. A primary culture of LN cells was preparedfrom mice immunized with 250 μg GA RS+CFA. The cells were cultured inthe presence of 2 different concentrations of GA RS and of GA DrugSubstance (DS) of different molecular weights. Following overnightincubation at 37° C. the culture media were collected and assayed forIL-2 by ELISA.

FIGS. 12(A & B): Cross-reactivity of GA RS-specific T cells with GA DSand Drug Product (DP) batches. A primary culture of LN cells wasprepared from mice immunized with 250 μg GA RS+CFA. The response of theGA RS-specific T cells to another GA DS batch (FIG. 12A), to a GA DPbatch and to mannitol (FIG. 12B) was compared to the response to the GARS batch. IL-2 levels in the culture media were measured by ELISA.

FIG. 13: Kinetics of GA RS proteolysis by trypsin. GA RS was proteolysedby trypsin for the indicated time points. The activity of theproteolysed samples was tested by the in vitro potency test.

FIG. 14: Reverse-Phase High Pressure Liquid Chromatography (RP-HPLC) ofGA before and after proteolysis by trypsin. GA RS was proteolysed bytrypsin and the chromatographic profile of the samples was tested byRP-HPLC.

FIG. 15: Kinetics of GA RS proteolysis by chymotrypsin. GA RS wasproteolysed by chymotrypsin for the indicated time points. The activityof the proteolysed samples was tested by the in vitro potency test.

FIG. 16: RP-HPLC of GA before and after proteolysis by chymotrypsin. GARS was proteolysed by chymotrypsin and the chromatographic profile ofthe samples was tested by RP-HPLC.

DETAILED DESCRIPTION

The subject invention provides a process for measuring the potency of atest batch of glatiramer acetate relative to the known potency of areference batch of glatiramer acetate which comprises

-   -   a. immunizing female (SJLXBALB/C)F1 mice between 8 and 12 weeks        of age with a predetermined amount of glatiramer acetate from        the reference batch.    -   b. preparing a primary culture of lymph node cells from the mice        of step (a) 9-11 days after immunization;    -   c. separately incubating at least five reference samples, each        of which contains a predetermined number of cells from the        primary culture of step (b) and a predetermined amount of        glatiramer acetate between 1 μg/ml and 25 μg/ml from a reference        batch;    -   d. incubating at least two samples, each of which contains a        predetermined number of cells from the primary culture of        step (b) and a predetermined amount of glatiramer acetate from        the test batch;    -   e. determining for each sample in steps (c) and (d), the amount        of interleukin-2 secreted by the cells in each sample after        18-21 hours of incubation of such sample;    -   f. correlating the amounts of interleukin-2 secreted by the        samples incubated with the test batch of glatiramer acetate with        the amounts of interleukin-2 secreted by the samples incubated        with the reference batch of glatiramer acetate so as to        determine the potency of the test batch of glatiramer acetate        relative to the reference batch of glatiramer acetate,    -   wherein in each sample in steps (c) and (d), the predetermined        number of cells is substantially identical, and wherein for each        sample containing a predetermined amount of glatiramer acetate        from the test batch there is a corresponding reference sample        containing a substantially identical predetermined amount of        glatiramer acetate from the reference batch.

In one embodiment, six reference samples are separately incubated instep (d).

The subject invention also provides a process for measuring the potencyof a test batch of glatiramer acetate relative to the known potency of areference batch of glatiramer acetate which comprises

-   -   a. immunizing a test mammal with a predetermined amount of        glatiramer acetate from the reference batch;    -   b. preparing a primary culture of cells from the test mammal of        step (a) at a predetermined time after immunization;    -   c. separately incubating at least two reference samples, each of        which contains a predetermined number of cells from the primary        culture of step (b) and a predetermined amount of glatiramer        acetate from a reference batch;    -   d. incubating at least two samples, each of which contains a        predetermined number of cells from the primary culture of        step (b) and a predetermined amount of glatiramer acetate from        the test batch;    -   e. determining for each sample in steps (c) and (d), the amount        of a cytokine secreted by the cells in each sample after a        predetermined time period of incubation of such sample;    -   f. correlating the amounts of the cytokine secreted by the        samples incubated with the test batch of glatiramer acetate with        the amounts of the cytokine secreted by the samples incubated        with the reference batch of glatiramer acetate so as to        determine the potency of the test batch of glatiramer acetate        relative to the reference batch of glatiramer acetate,    -   wherein in each sample in steps (c) and (d), the predetermined        number of cells is substantially identical, and wherein for each        immunization sample containing a predetermined amount of        glatiramer acetate from the test batch there is a corresponding        reference sample containing a substantially identical        predetermined amount of glatiramer acetate from the reference        batch.

In one embodiment, the cytokine is an interleukin.

In a preferred embodiment, the interleukin is interleukin-2.

In another embodiment, the interleukin is interleukin-6.

In a further embodiment, the interleukin is interleukin-10.

In an added embodiment, the cytokine is interferon-gamma.

In one embodiment, the mammal produces T cells specific to glatirameracetate reference standard.

In another embodiment, the mammal is a rodent.

In still another embodiment, the rodent is a mouse.

In an additional embodiment, the mouse is a female (SJLXBALB/C)F1 mouse.

In a further embodiment, the mammal is about 8 to about 12 weeks old.

In yet another embodiment, the cells are lymph node cells.

In one embodiment, the cells are spleen cells.

The subject invention further provides a process for preparing a batchof glatiramer acetate as acceptable for pharmaceutical use whichcomprises

-   -   a. preparing a batch of glatiramer acetate;    -   b. measuring the relative potency of the batch according to the        process of claim 1; and    -   c. qualifying the batch as acceptable for pharmaceutical use if        the relative potency so measured is between 80% and 125% of the        reference batch of glatiramer acetate.

Additionally, the subject invention provides a process for preparingglatiramer acetate acceptable for pharmaceutical use which comprises

-   -   a. preparing a batch of glatiramer acetate;    -   b. measuring the relative potency of the batch according to the        process of claim 3; and    -   c. qualifying the batch as acceptable for pharmaceutical use if        the relative potency so measured is between SOW and 125% of the        reference batch of glatiramer acetate.

Thus, rte present invention provides the standardization of themeasurement of the potency of GA. The potency test quantitativelydetermines the biological activity of GA. This is the first showing everof such a test. This standardization method is essential in order toshow batch to batch reproducibility with regards to potency and qualityof DS and DP. In the context of this application, OS refers to theactive ingredient, i.e., GA. DP is used to indicate the finishedproduct, i.e., Copaxone®. RS denotes a batch of glatiramer acetatehaving an average molecular weight of about 7000 Da.

The subject invention makes use of the observation that T cellsincubated with a cytokine, e.g., IL-2, proliferate in response to thatcytokine (Lisak et al., 1974).

The examples which follow describe the invention in detail with respectto showing how certain specific representative embodiments thereof canbe made, the materials, apparatus and process steps being understood asexamples that are intended to be illustrative only. In particular, theinvention is not intended to be limited to the methods, materials,conditions, process parameters, apparatus and the like specificallyrecited herein.

EXPERIMENTAL EXAMPLES General Procedure Outline

Mice were immunized with 250 μg GA RS in CFA. GA RS was produced asdescribed in U.S. Pat. No. 5,800,808 or PCT International PublicationNo. WO 00/05250. The GA RS was chosen based on the chemical andbiological properties being in the midrange of Copaxone® as describedabove. After 9-11 days, a primary culture of LN cells was prepared, andthe cells were incubated with various concentrations of GA RS and withtest samples. Following 18-21 hours of incubation at 37° C. in ahumidified CO₂ incubator, the culture media were collected and the levelof IL-2 was measured by ELISA. The T-cell response to each DS batch weretested at two concentrations (within the linear range), and the %potency of the DS batch was calculated relative to that of the GA RSbatch.

Example 1 Standard Procedure Purpose

The purpose of this procedure was to determine the relative potency ofGA DS batch in vitro, using GA RS-specific T cells.

Equipment

Laminar hood, hemacytometer, disposable cover slips, cell countercentrifuge, temperature-controlled shaking incubator, humidified,temperature controlled 5% CO₂ incubator, light and inverted microscopes,ELISA reader (450 nm filter), freezer, refrigerator scissors, forceps,stepper, pipettman 40-200 μl, pipettman 200-1000 μl, pipettman 5-40 μl,powerpette, sterile glass syringes and luer bridges.

Disposables

Cryotubes, 96-well enhanced binding ELISA plate (Nunc, Cat. #442404),96-well non-sterile microtest plate (Falcon Cat. #3911), 24-well flatbottom steriled tissue culture plate (Nunc, Cat. #143982), petri dishes,Eppendorf tubes (polypropylene), steriled pipette tips 200-1000 μl,pipettes: 2, 5 & 10 ml, laboratory coat, gloves, 0.2μ cellulose acetatefilter, filtered system 200 ml (Corning, Cat. #430767), Kim wipes,support platform, 10 ml syringes, 21Gxl 1/2″ needles, insulin syringesand combitips 5 ml.

Materials and Reagents For the Immunization Procedure

95% ethanol (Bio Lab, Cat. #13680605, or equivalent), 70% ethanolprepared from 95% ethanol by dilution with distilled water, phosphatebuffered saline (PBS)×1 (SIGMA, Cat. #3813, or equivalent), CFAcontaining 1 mg mycobacterium tuberculosis (MT) (H37Ra, ATCC 255177),(SIGMA, Cat. #F-5881, or equivalent), and GA RS batch.

For the In Vitro Bioassay Procedure

95% ethanol (Bio Lab, Cat. #13680605, or equivalent), 70% ethanolprepared from 95% ethanol by dilution with distilled water, trypan blue(BDH, Cat. #3407), DCCM1 (Defined Cell Culture Media) (Beit Haemek, Cat.#05-010-1A or equivalent), RPMI 1640 (Roswell Park Memorial Institute)(Beit Haemek, Cat. #01-100-1A), steriled L-glutamine 2 mM×100 (Bio Lab,Cat. #13.015), steriled MEM (Minimum Essential Media)—non-essentialamino acids×100 (Bio Lab, Cat. #11.080), steriled sodium pyruvate 1mM×100 (Bio Lab, Cat. #13.016), antibiotic/antimycotic Solution 1 (BioLab, Cat. #13.020), 2-mercaptoethanol (SIGMA, Cat. #M-7154), PBS (SIGMA,Cat. #3813), concavalin A (Con A) (SIGMA, Cat. #C-5275), MBP (MyelinBasic Protein) peptide (87-99) (BACHEM, Cat. #H-1964, or equivalent),and GA RS.

IL-2 was measured by ELISA kit: OptEIA™ Set: mouse IL-2 (Pharmingen,Cat. #2614KI, or equivalent).

Animals

Female (SJLXBALB/C)F1 mice between 8-12 weeks old (Jackson Laboratories,Bar Harbor, Me.) were used, although female (BALB/C)F1 mice between 8-12weeks old from other sources may be used. Animal housing and careconditions were maintained in specific pathogen-free (SPF) conditions.

Solutions

TABLE 1 Procedures for making solutions Steriled PBS The content of onepackage of PBS was dissolved in 1 liter double distilled water (ddH₂O).The buffer was filtered through a 0.2μ cellulose acetate filter and keptin refrigerator (2-8° C.) up to one week. 2% (w/v) Trypan blue in About0.5 g of Trypan blue were dissolved in 25 ml PBS filtered PBS and storedin refrigerator up to 6 months. 0.1% (v/v) Trypan blue in 0.1% Trypanblue solution was prepared from the 2% PBS Trypan blue stock solutionand filtered through a 0.2μ cellulose acetate filter. The 0.1% Trypanblue solution was stored at room temperature for up to one month.Steriled 2- 10 μl of 2-mercaptoethanol were added into 9.99 mlMercaptoethanol of sterilized PBS and filtered through a 0.2μ celluloseacetate membrane and kept in refrigerator for up to 3 months. GA RSAbout 10 mg of glatiramer acetate RS were weighed stock solution ofaccurately and dissolved in ddH₂O to a concentration 1 mg/ml of approxand 1.2 mg/ml. The optical density (OD) of the RS solution was measuredat 275 nm. The OD was adjusted to approx. 1.03 with ddH₂O, and a stocksolution of 1 mg/ml of GA RS was obtained. The solution was mixed well,divided into working aliquots (200-500 μl) and stored at −20° C. untiluse. Steriled MEM × 100 The steriled solution was divided into aliquotsof 5 ml each, and kept at −20° C. until use. After thawing, the workingaliquot was kept in refrigerator for up to one month. Antibiotic/ Theoriginal 20 ml package was kept at 20° C. The antimycotic package wasopened and all the contents were solution 1 divided into aliquots of 2ml each and stored at −20° C. until use. The working aliquot was stableand was able to be subjected to several freeze-thaw cycles. Steriled Thecontents of the opened package were divided L-glutamine into aliquots of2 ml each and kept at −20° C. until 2 mM × 100 use. Enriched DCCM1 For100 ml of sterile enriched DCCM1, the following medium components weremixed together: 1 ml of L-glutamine (2 mM), 1 ml MEM, 1 ml sodiumpyruvate (1 mM), 200 μl antibiotic/antimycotic solution 1, 400 μl2-mercaptoethanol and 96.4 ml DCCM1. The enriched DCCM1 was filteredthrough a 0.2μ cellulose acetate filter and stored in refrigerator forup to 1 week. MBP peptide Primary bank: The stock solution of 10 mg/mlin ddH₂O was prepared, divided into working aliquots and kept at 20° C.Secondary bank: One aliquot of the primary bank (10 mg/ml) was thawedand diluted to 1 mg/ml with ddH₂O. The primary bank solution was dividedinto working aliquots of 50 μl each and kept at −20° C. Upon use,aliquot from the secondary bank was thawed and diluted with enrichedDCCM1 to obtain a solution of 20 μg/ml. Con A solution Primary bank: Thecontents of one vial of 5 mg Con were dissolved in 1 ml PBS, mixed welland divided into aliquots of 50 μl each. The aliquots were kept at −20°C. up to the expiration date set by the manufacturer. Secondary bank:One aliquot of the primary bank (5 mg/ml) was thawed and diluted with4.95 ml of enriched DCCM1 medium to obtain a solution of 50 μg/ml. Thesolution was divided into working aliquots of 100 μl each and kept at−20° C. Upon use, one aliquot from the secondary bank was thawed anddiluted up to 1 ml (a 10-fold dilution) with enriched DCCM1 to obtain asolution of 5 μg/ml Con A.

Immunization

GA RS emulsion in CFA was prepared under sterile conditions, i.e., in alaminar hood, using sterile equipment and materials.

Preparation of GA RS Solution

About 15 mg of GA RS were weighed accurately and dissolved in sterilePBS to a concentration of 5 mg/ml.

Preparation of GA RS Emulsion

Equal volumes of GA RS solution (5 mg/ml) and CFA were mixed. Themixture was transferred into a sterile glass syringe connected to asecond glass syringe through a luer bridge. The mixture was mixed wellby being transferred from one syringe to another until the mixture waswell emulsified. A stable emulsion was confirmed when a drop of theemulsion floated on water without dispersing.

Injection

The GA RS emulsion was transferred into an insulin syringe. Then, 100 μlof the emulsion (250 μg GA per mouse) were injected into four footpadsof each naive mouse (about 25 μl into each footpad). The immunized micewere used for the in vitro test 9-11 days following immunization.

Preparation of a Primary Culture of LN Cells

The primary culture of LN cells was prepared 9-11 days followingimmunization, according to the following procedure:

Surgical procedure for Removal of LN Cells

The UV lamp was turned on 20 minutes before commencing work in thelaminar hood and turned off when work began. Prior to placing anyreagents under the hood, the working surface was cleaned with a 70%ethanol solution. Enriched DCCM1 medium was prepared. The enriched DCCM1and the RPMI medium were pre-warmed at 37° C. prior to use. The micewere sacrificed by cervical dislocation. Each mouse was placed on itsback and fastened to a support platform. The abdomen was sprayed with70% alcohol and a middle incision was made (a 2 cm-long incision wasusually sufficient). The skin was intersected towards the hind legs andLN was located from the hind and forelegs. The LN was transferred into asterile petri dish containing about 5 ml sterile RPMI medium and the LNcells were teased out by a sterile syringe plunger. The sterile syringewas used to collect the cells' suspension from the petri dish (thecollection of tissue debris was avoided by using sterile needles). Thecells' suspension were transferred into a 50 ml sterile tube.

Cell Counting: Example Procedure for Counting LN Cells Derived from 5Immunized Mice

The cells' tube were filled with RPMI medium up to 40 ml. The LN cellswere centrifuged at 200×g for 10 minutes at room temperature (15-25°C.). The pellet was re-suspended with 40 ml RPMI. Two aliquots of 50 μlwere drawn each from the cells' suspension diluted 4-fold with 150 μl of0.1% Trypan blue in a microtest well. The aliquots were mixed well bypipetting gently up and down. The hemacytometer was covered with a coverslip. A 50-200 μl pipettman was used to load both aliquots into theupper and lower chambers of the hemacytometer, one suspension in eachchamber. The mixture was allowed to settle within the chambers for about2 minutes. Care was taken to not introduce bubbles into the chamber. Themixture (cell suspension+Trypan blue) was allowed to cover the entiresurface of the chamber. If bubbles were present in the chamber, or if itwas overloaded, the hemacytometer was cleaned completely and dried withwipes and the chambers were reloaded.

The viable cells were counted in the central square (composed of 25large squares of 16 small squares each, see FIG. 3) of the upper andlower chambers. The viable cells did not absorb Trypan blue and weretherefore characterized by a clear appearance. However, dead cells werepermeable to Trypan blue and appeared blue in color. Cells appearing onthe border of the central square were only counted if a portion of thecell was actually within the central square. If a cell was on theborder, and not at all within the central square, it was not counted.The cells' density was calculated using the following equation:

${{Average}\mspace{14mu} {number}\mspace{14mu} {viable}\mspace{14mu} {cells}\mspace{14mu} \left( {{both}\mspace{14mu} {chambers}} \right) \times 4 \times 10^{4}} = {\frac{cells}{ml}.}$

The cells were centrifuged at 200×g for 10 minutes at room temperature.The cells were re-suspended to a density of 1×10⁷ cells/ml with enrichedDCCM1.

In Vitro Bioassay

The in vitro bioassay was performed in a 24-well, flat-bottomed tissueculture test plate at a final volume of 1 ml.

Preparation of GA RS Calibration Curve

One aliquot of the 1 mg/ml GA RS stock was thawed. The GA RS stocksolution was diluted to 100 μg/ml (10-fold) with enriched DCCM1 mediumand filtered through a 0.2μ cellulose acetate filter. Six serialdilutions of the GA RS solution with enriched DCCM1 medium were preparedbetween 2-50 μg/ml, as described by the example in Table 2.

TABLE 2 Example for preparation of GA RS dilutions GA RS VOLUME (μl) OFGA RS VOLUME CONCENTRATION STOCK SOLUTION (μl) OF ENRICHED (μg/ml) (100μg/ml) DCCM1 50 1000 1000 30 600 1400 20 400 1600 10 200 1800 5 100 19002 40 1960

Preparation of GA DS Dilutions

About 10-20 mg of GA DS from the batch to be tested was weighedaccurately and dissolved with ddH₂O to 1.2 mg/ml. The OD minus blank ofthe solution was measured at 275 nm. The OD of the sample was adjustedto approx. 1.03 with ddH₂O to obtain a stock solution of 1 mg/ml of GA.The stock solution of 100 μg/ml was prepared with enriched DCCM1 andfiltered through a 0.2μ cellulose acetate filter. The stock solution wasdiluted to 10 and 20 μg/ml as described in Table 2 for the RS batch.

Assay Reaction

The following were added to the 24-well flat-bottomed tissue cultureplate (see an example of a plate template below):

GA RS

0.5 ml of LN cells (final density, for example, 5×10⁶ cells/well).

0.5 ml of each GA RS dilution, thus the final concentrations of GA RS inthe wells were 25, 15, 10, 5, 2.5 and 1 μg/ml.

GA DS samples

0.5 ml of LN cells (final density, for example, 5×10⁶ cells/well).

0.5 ml of each sample dilution, thus the final concentrations of thetest sample in the well were 5 and 10 μg/ml.

Each test included the following controls:

-   -   1) Negative control—LN cells incubated with a control peptide:        -   0.5 ml of LN cells (final density 5×10⁶ cells/well)        -   0.5 ml of MBP peptide solution (20 μg/ml) in enriched        -   DCCM1 (final concentration 10 μg/ml)    -   2) Positive control—LN cells stimulated with Con A (non-specific        T cell stimulant):        -   0.5 ml of LN cells (final density 5×10⁶ cells/well)        -   0.5 ml of Con A (5 μg/ml) in enriched DCCM1 (final            concentration 2.5 μg/ml)

Example for a Plate Template

GA RS* GA RS* GA RS* GA RS* GA RS* GA RS* 1 μg/ml 2.5 μg/ml 5 μg/ml 10μg/ml 15 μg/ml 25 μg/ml Sample 1 Sample 1 5 μg/ml 10 μg/ml Sample 2Sample 2 5 μg/ml 10 μg/ml Sample 3 Sample 3 Negative Positive 5 μg/ml 10μg/ml Control Control GA RS*—Glatiramer acetate reference standard.

The density of the cells was changed depending upon their response toGA. The cultures were kept at 37° C. in a humidified 5% CO₂ incubatorfor 18-21 hrs. The plate was centrifuged at 200×g for 10 minutes at roomtemperature. The supernatants were collected into cryotubes. Thesupernatants were divided into working aliquots to avoid repeatedfreezing/thawing of the samples. The supernatants were stored at −20° C.for up to one week. The hood was cleaned with 70% ethanol solution anddried with Kim wipes. The gloves were removed and the hands wereimmediately washed with disinfectant.

ELISA for IL-2 Detection

All samples were tested in triplicate. Each plate run included thefollowing:

-   -   1) IL-2 standard curve—including at least 6 non-zero        concentrations of IL-2.    -   2) Blank    -   +1^(st) antibody, without IL-2 standard, +2^(nd) antibody (zero        point).    -   3) Samples    -   The culture media of GA RS, test samples and controls were        diluted with enriched DCCM1 as follows:        -   a) A 2-fold dilution of the 1 and 2.5 μg/ml GA RS sample and            of the negative control sample;        -   b) 5-10 fold dilutions of the 5-25 μg/ml GA RS samples and            of the test samples; and        -   c) 15-20 fold dilution of the positive control sample (Con            A).

The ELISA protocol for measuring IL-2 levels was performed according tothe manufacturer's recommendations. If the optical density of any samplereached the upper/lower limits of the plate reader, the sample wasre-analyzed at a higher/lower dilution, respectively.

Calculation, and Acceptance Criteria ELISA Measurements

The mean absorbance was subtracted of the blank sample (zero IL-2standard point) from the absorbance of standards, samples and controlsand calculated for each set of triplicate the mean (absorbance-blank),standard deviation (SD), and relative standard deviation (RSD).

Sample Replicates

Whenever there was a suspected outlier, it was necessary to ensure thatthe outlier was statistically based, in order to elucidate any potentialproblems that may have affected the overall results. If the RSD betweentriplicate measures was higher than 10% and the average OD-blankwas >0.300, outlier rejection was applied using the Dixon Q-Test, TheDixon Q-Test was used to reject possible outliers when the relevantacceptance criteria was not satisfied in a test based on replicates. Theoutlier test was applicable only to replicate measurements of the samestandard solution. For less than 10 observations, only 1 outlier wasable to be determined and eliminated. This procedure expanded the use ofthe Dixon Q-Test in rejecting outliers from any number of replicatemeasurements between 3 and 7, with a confidence level of 95%.

Procedure

The suspected outlier was designated X₁. All other measurements werelabeled in reference to the suspected outlier, e.g., X₂ was the valuenext to the suspected outlier, X₃ was second value from the suspectedoutlier, X_(k) was the farthest from the suspected outlier and X_(k-1)was the value second from the farthest, etc. For 3-7 replicates, thefollowing equation was used:

$\frac{X_{2} - X_{1}}{X_{k} - X_{1}}.$

The appropriate k value was determined from the calculated fractionusing Table 3.

TABLE 3 k Value No. of Observations (k) Value at P₉₅ 3 0.94 4 0.76 50.642 6 0.560 7 0.507

If no outlier was identified by the Dixon Q-Test but the % differencebetween 2 out of the 3 replicates was not more than 10%, the closest 2replicates were used for calculating the % potency. Otherwise, the ELISAtest was repeated for this sample.

Outlier rejection from samples with OD<0.300 (blank, negative controland low standard points) was applied. When an outlier was located, whenit was rejected and reported. Duplicate measures were used for thecalculation of % potency.

Blank Samples

The absorbance of each of the blank samples was ≦10% of the meanabsorbance of the highest concentration of the IL-2 standard. If one ofthe blank replicates was beyond the above limits, it was rejected andduplicate samples were used.

IL-2 Standard Curve

The IL-2 standard curve was graphed according to the manufacturer'srecommendations. IL-2 standards that exhibit poor sensitivity, or sampleprocessing error were able to be rejected if a minimum of six non-zeroconcentration IL-2 standards remained in the curve. The back-calculatedstandard concentration had a relative error (RE) greater than 20% forthe lower calibration point and ±15% for all other concentrations. TheIL-2 calibration curve was constructed from at least six non-zeroconcentration points (at least 17 calibration points), covering therange of expected concentrations. The standard curve range was able tobe truncated if the high or low concentrations failed. The R² of thelinear regression curve was 0.97.

Assay Controls

The concentration of IL-2 was calculated in all samples from the linearregression plot of the IL-2 standard, utilizing the equation of thelinear regression curve. The final concentration of IL-2 was calculatedin all samples by multiplying by the samples' dilution factor.

Negative Control (MBP Peptide)

The final concentration of IL-2 in at least 2 out of the 3 replicates ofthe negative control sample was below the levels of IL-2 measured forthe lowest calibration point of the GA RS curve.

Positive Control (Con A)

The final concentration of IL-2 in at least 2 out of the 3 replicates ofthe positive control sample was similar to or above the level of IL-2 inthe highest calibration point of the GA RS curve.

Calculation of the Relative Potency of GA DS Batches GA RS Curve

The GA RS curve was plotted on a log-log scale, with log IL-2concentration on the y-axis and log GA RS concentration on the x-axis.The calibration curve was constructed from at least five non-zeroconcentrations (at least 14 calibration points). Calibration points wererejected as described for the IL-2 standard points. The best-fitregression curve was computed through the standard points. The R² was≧0.97. The slope (β) was ≧0.77.

Parallelism Analysis

The dose-response curve of each test sample was plotted on a log-logscale, with log IL-2 concentration on the y-axis and log GA DSconcentration on the x-axis. The best fit regression curve was computedthrough the sample points. The slope (β*) was within the followingrange:

β×0.635

β*

β×1.365.

If β* was out of limits, the in-vitro test was repeated in duplicate(two separate sample preparations). If β* in one re-test failed, thebatch was rejected. If β* in both re-tests was within limits, the %potency and 95% fiducial limits were determined.

Estimation of the % Potency and the Fiducial Limits

The estimate of the random error to be used to determine the FiducialLimits (which have a 95% probability of including the “true % potency”)was obtained by using ANOVA. This statistical technique splits the totalvariation between observed responses into separate components, namely:

1. due to linear dose-response {close oversize brace} Model 2. due tothe mean effect of preparation 3. due to deviation from parallelism 4.due to deviation from linearity {close oversize brace} Random 5. due toresidual between-replicate variation Error

The components 3 and 4 were included in the random error term due tonon-significant deviations from linearity and parallelism, respectively.The total sum of squares was partitioned into 3 components(SS-Regression, SS-Preparation and SS-Error), the appropriate number ofdegrees of freedom and the F-test for significance.

The % potency of the tested batch was calculated and the 95% fiduciallimits for the estimated potency as described below:

Computational Algorithm for the Calculation of Relative Potency and 95%Fiducial Limits

-   -   Step 1: Compute the transformation of the given data (GA. Batch        and GA. RS.) Into log₁₀ scale:

Y_(id)=log₁₀(response_(id)); i=1, . . . n _(k)

X_(id)=log₁₀(dose_(id)); i=1, . . . n _(k)

where k=1,2 is a preparation Index of GA, batch and GA, RS,respectively;n₁ end n₂ are the total numbers of measurements performed for the GA,batch and GA, RS, respectively.

Thus, N=n₁+n₂ is an overall total number of observations.

-   -   Step 2: Calculate the common slope for the linear regression        based on all measured data points vial the formula:

${\beta = \frac{\sum\limits_{j = 1}^{N}\; {\left( {Y_{j} - \overset{\_}{Y}} \right) \cdot \left( {X_{j} - \overset{\_}{X}} \right)}}{\sum\limits_{j = 1}^{N}\; \left( {X_{j} - \overset{\_}{X}} \right)^{2}}};$

-   -   where Y is an overall mean value of log₁₀(response);    -   X is an overall mean value of log₁₀(dose).    -   Step 3: Calculate the sum of squares due to regression on        log₁₀(dose) as:

${{SS}_{REG} = {\beta^{2} \cdot {\sum\limits_{j = 1}^{N}\; \left( {X_{j}^{i} - \overset{\_}{X}} \right)^{2}}}};$

-   -   Step 4: Calculate the random error sum of squares as:

${{SS}_{ERR} = {\sum\limits_{k = 1}^{2}\; {\sum\limits_{i = 1}^{n_{k}}\; \left\lbrack {Y_{ki} - {\overset{\_}{Y}}_{k} - {\beta \cdot \left( {X_{ki} - {\overset{\_}{X}}_{k}} \right)}} \right\rbrack^{2}}}};$

where Y_(k) , X_(k) are mean log₁₀(response) and log₁₀(dose) values,respectively, of preparation k.

-   -   Step 5: Calculate the Mean Square Error term as following:

DF_(ERR)(random error degrees of freedom)=N−3;

MS_(ERR)=SS_(ERR)/DF_(ERR).

-   -   Step 6: Use the statistical tables of t-distribution in order to        find the appropriate value of t-statistic:

t=t(0.975,DF_(ERR)).

-   -   Step 7: Calculate the point estimate of the relative potency as        following:

${{\% \mspace{14mu} {Potency}} = {{10\frac{{\overset{\_}{Y}}_{1} - {\overset{\_}{Y}}_{2}}{\beta}} - {{\left( {{\overset{\_}{X}}_{1} - {\overset{\_}{X}}_{2}} \right) \cdot 100}\%}}};$

-   -   Step 8: Calculate the expression denoted by C via the formula:

${C = \frac{{SS}_{REG}}{{SS}_{REG} - {{MS}_{ERR} \cdot t^{2}}}};$

-   -   Step 9: Calculate the logarithms of lower and upper limits of        95% Fiducial Interval:

${{{Log}_{10}\left( {{Lower}\mspace{14mu} {Limit}} \right)} = {{C \cdot \frac{{\overset{\_}{Y}}_{1} - {\overset{\_}{Y}}_{2}}{\beta}} - \left( {{\overset{\_}{X}}_{1} - {\overset{\_}{X}}_{2}} \right) - {\frac{\sqrt{{MS}_{ERR} \cdot C} \cdot t}{\beta} \cdot \sqrt{\frac{1}{n_{1}} + \frac{1}{n_{2}} + \frac{\left( {{\overset{\_}{Y}}_{1} - {\overset{\_}{Y}}_{2}} \right)^{2}}{{SS}_{REG} - {{MS}_{ERR} \cdot t^{2}}}}}}};$${{{Log}_{10}\left( {{Upper}\mspace{14mu} {Limit}} \right)} = {{C \cdot \frac{{\overset{\_}{Y}}_{1} - {\overset{\_}{Y}}_{2}}{\beta}} - \left( {{\overset{\_}{X}}_{1} - {\overset{\_}{X}}_{2}} \right) + {\frac{\sqrt{{MS}_{ERR} \cdot C} \cdot t}{\beta} \cdot \sqrt{\frac{1}{n_{1}} + \frac{1}{n_{2}} + \frac{\left( {{\overset{\_}{Y}}_{1} - {\overset{\_}{Y}}_{2}} \right)^{2}}{{SS}_{REG} - {{MS}_{ERR} \cdot t^{2}}}}}}};$

Step 10: Transform the values computed in the previous step intooriginal scale by taking of anti-logarithms of the resulting log-limitsand multiply by 100%.

The estimated potency of GA DS batch was not less than 80% and not morethan 125% of the stated potency. The fiducial limits of error (P=0.95)of the estimated potency were less than 70% and not more than 143% ofthe stated potency. If the batch was outside the above limits, thein-vitro test was repeated in duplicate. If the results of both re-testswere within specifications, the batch was acceptable. If one re-testfailed, the batch was rejected.

Documentation

The LN cell count and ELISA plates template were recorded. The originalELISA reader records and the result form were filed.

Example 2 Development of Standard Procedure of Example 1

Experiment 2A: Profile of Cytokines Secreted from GA RS-Specific T Cells

The LN cells were derived from female (SJL×BALB/C)F1 mice immunized with250 μg GA RS in CFA 9-11 days earlier were cultured in the presence ofvarious concentrations of GA RS. The cells were incubated with GA RS for18-24 hours at 37° C. in a 5% CO₂ humidified incubator. Subsequently,the cultures were centrifuged and the supernatants collected and assayedfor cytokines by ELISA.

The ELISA was performed using biotinylated antibodies specific to thecytokine and strepavidin-horseradish peroxidase (HRP) conjugated fordetection. Each plate ran included blank control (first and secondantibodies without the cytokine standard). Each plate ran also includedquality control (QC) samples (three concentrations of cytokine standardwithin the assay's linear range). Each in vitro test included a positivecontrol (Con A, a non-specific T-cell stimulant) and a negative control(no GA or any other antigen). All the cytokines were measured after18-24 hours of incubation. Levels of TGF-β, IL-10 and IL-4 were testedagain after 72 hours of incubation. The results are shown in Table 4.

TABLE 4 Cytokine profile Cytokine Secretion levels IL-2 ++ INF-γ ++IL-6 + L-10 + L-13 − TGF-β − IL-4 − TNF-α −

In Table 4, the maximal levels measured for each cytokine are presentedin arbitrary units: (−) detection limit; (+) up to −400 pg/ml; and(++)>400 pg/ml. Table 4 shows that in response to GA RS in culture, theLN cells secreted IL-2, INF-γ, IL-10 and IL-6, while TNF-α, IL-4, IL-13and TGF-β were not detected in the culture media. These results indicatethat the cytokines produced by the GA RS-specific T cells are of Th₀type. It should be noted that a Th₀ profile was observed in differentimmunization protocols, i.e., immunization with IFA or with low doses ofGA.

Since IL-2 is a good marker for T cell activation, and since thesecretion of IL-2 in response to GA RS was very reproducible, with alinear dose-response relationship, IL-2 seemed to be the optimumcytokine to measure T cell activation.

Experiment 2B: Optimization of the Immunization Procedure

Several experiments were performed to establish the optimal immunizationprotocol. The first experiment tested the effect of GA RS (immunizingantigen) dose on T-cell responses in the LN and in the spleen. Twogroups of 10 mice each were immunized with either 250 μg GA in CFA(group 1) (as in the EAE blocking test) or with 10 μg GA in CFA (group2). Primary cultures were prepared from both the LN and the spleens ofthe immunized mice. The cultures were incubated overnight with variousdoses of GA RS and afterwards the culture media were collected andassayed for IL-2 as in Experiment 2A. The results in FIG. 4 clearly showthat in both immunization protocols the levels of IL-2 secreted from LNcells are high compared to those secreted from spleen cells. Based onthese results it was decided to use primary cultures of LN cells for theassay. In addition, the doses of GA RS injected into mice did not affectthe T cell response in culture. Both LN and spleen cells secretedsimilar quantities of IL-2 regardless of the immunizing dose of GA RS.This indicates that the immunization procedure is robust, and that evenmajor variations in the immunizing dose of GA RS do not affect theimmunological outcome.

For further optimization of the immunization protocol, one group wasinjected with 250 μg GA RS in CFA and the second group with 10 mg GA inICFA. The dose of GA RS in the second group was higher since ICFA, aweaker adjuvant, was used. Ten days later, the response of the LN cellsfrom both groups to GA RS was tested in vitro. FIG. 5 shows thatimmunization with 250 μg GA RS in CFA induced a much stronger responsein culture, although a much lower dose of antigen was used.

Based on these findings, and on the fact that 250 μg/mouse of GA in CFAis very effective in blocking EAE (at least 80% blocking of EAE in thismouse strain), 250 μg/mouse of GA in CFA appears to be the optimum dose.

Specific T cells were usually generated within approximately 10 days,following a single immunization with CFA. FIG. 6 shows the response ofthe LN cells to GA RS in culture, prepared 9, 10 and 11 days followingimmunization. Since the dose-response of IL-2 secretion was similar onall days, the immunization period may last for 9-11 days.

Experiment 2C: Optimization of the In Vitro Test Conditions

Several experiments were performed to establish the optimal protocol forthe in-vitro reaction. These studies included optimization of cultureconditions, incubation time, stability of IL-2 in test samples andstability of GA RS at −20° C.

i) Culture Media

Cultures of mouse lymphoid cells are usually maintained in RPMI medium,supplemented with 1% normal mouse serum. Normal mouse serum may containendogenous IL-2 that can be detected by the anti mouse IL-2 monoclonalantibodies used in the ELISA kit. In addition, the use of different lotsof normal serum may increase the inter-day variations of the in vitrotest. To avoid cross-contamination with endogenous mouse IL-2, and toreduce the inter-day variations of the method, the responses of theGA-specific T cells were tested in 4 different culture media: 1) RPMI+1%normal mouse serum (NMS); 2) RPMI+1% fetal bovine sera (FBS) (bovineIL-2 is not recognized by the anti mouse IL-2 used in the ELISA kit); 3)Biotarget (serum-free media produced exclusively by BeitHaemek, Israel);and 4) DCCM1 (serum-free media produced by various manufacturers).

FIG. 7 shows the results of a representative experiment that comparesthe response of LN cells to GA RS in different culture media. The bestresponses were observed when serum-free media were used. Thedose-response range was left-shifted in the absence of serum. This canbe explained by previous studies showing that GA binds to albumin and toother serum proteins. This binding may reduce the availability of GA inculture to interactions with APCs, and thus higher concentrations of GAare required to stimulate the T cells. Based on these results, theoptimum medium seems to be DCCM-1 medium.

ii) Kinetics of IL-2 Secretion.

IL-2 is an autocrine and paracrine growth factor that is essential forclonal T-cell proliferation and for functional properties of B cells andmacrophages. Following stimulation of the culture with GA RS, IL-2 issecreted by the activated GA-specific T cells and is subsequentlyconsumed by the LN cells. Kinetic studies of IL-2 secretion wereperformed in an attempt to determine the optimal (peak) time forcollection of the supernatants, following stimulation with GA. LN cellswere cultured and incubated with various concentrations of GA RS at 37°C. in a humidified CO₂ incubator. At the intervals indicated in FIG. 8,aliquots were sampled and the cells were removed by centrifugation. Thesupernatants were kept at 20° C. and at the end of the experiment wereassayed for IL-2 by ELISA.

FIG. 8 shows that the peak of IL-2 levels in the culture media isbetween 18-21 hours. The reduction in IL-2 levels in samples collectedfrom 24-48 hours can be explained by the consumption of IL-2 by the LNcells. Thus, the optimum time for supernatant collection appears to beafter 18-21 hours of incubation.

iii) Measurement of Cytokines

The method relies on accurate measurements of IL-2 in samples of GA RSand test samples. During the experiments, the levels of IL-2 weremeasured by OptEIA (Pharmingen, Cat. #2614KI)—an ELISA kit specific formouse IL-2. This ELISA kit is very sensitive and the results areaccurate and reproducible.

iv) Stability of IL-2 in test samples at −20° C.

In most of the experiments performed, the culture media were collectedand kept at −20° C. before being analyzed by the ELISA. Preliminarystudies of the stability of IL-2 in culture media show that the cytokineis stable for one week at −20° C. (FIG. 9). Therefore, the culture mediaof the in-vitro test samples can be kept at −20° C. for up to one weekprior to measuring IL-2. The results of this experiment alsodemonstrated that the ELISA results are very reproducible—the levels ofIL-2 measured in the samples were practically identical in two ELISAplate runs performed on two different days one week apart.

v) Stability of GA RS Solution at −20° C.

To test the stability of GA RS solution at −20° C., the dose-response ofa GA RS solution was tested immediately following preparation, and afterstorage for 5 months at −20° C. FIG. 10 shows that there is practicallyno difference in the dose-response curves of GA RS solution before andafter storage for 5 months at −20° C. Therefore, aliquots of GA RSsolution can be prepared and kept at −20° C. for at least 5 monthsbefore use.

Experiment 2D: Determination of Linear Range of GA RS Calibration Curves

The statistical validation was carried out based on GA RS calibrationcurves calculated and evaluated separately for each one out of 21 platesreceived for the analysis. These 21 samples were gathered at differenttimes over an approximately four-month period. The GA concentrationrange for the given plates varied from 0.25 to 50 μg/ml. The followingvalidation characteristics derived from the GA RS calibration curvesconstituted the main concern of the analysis:

-   1. Optimal transformation to ensure wider limits of the linear    range;-   2. Determination of the linear range limits;-   3. Overall criteria for accepting a calibration curve;-   4. Estimation of assay accuracy and precision;-   5. Assessment of duplicate reliability (see the paragraph below).

The nature of the experiments was such that there were typically 3replicates (triplicates) at each calibration point. However, in someinstances, when a triplicate measurement could not be provided, theassessment of duplicate reliability became essential.

Linearity of GA Dose-Response Relationship

The basis of most aspects of the validation discussion presented belowwas a linear regression model that related the IL-2 concentration(pg/ml) to the GA concentration (μg/ml). The assumption of the linearityof this relationship was necessary for the appropriate fitting of thelinear regression model. The data was plotted in a Linear-Linear scale.The same relationship was transformed into Log-Log scale, as well as aLog-Linear scale, and a Log-Square Root scale. The Log-Logtransformation demonstrated the most suitable linear features. Thus, thechosen form of the regression model was the Log-Log one:

Log₁₀(IL-2 conc)=a+β*Log₁₀(GA conc)+error

The response variable was a log-transformed mean of the 3 replicatesmeasured at each calibration point. This model was fitted to eachcalibration sample and the appropriate statistics (R², intercept, andslope) were calculated for each fitted curve. The value of R² reflectedthe ratio of the residual sum of squares (RSS) to the total sum ofsquares (TSS) via the formula:

R ²=1−RSS/TSS

Linear Range Determination

The linear range was determined based on the following criteria:

-   1. Visual inspection of plotted log₁₀ (IL-2 conc) vs. log₁₀ (GA    conc);-   2. The regression influence diagnostics, such as Cook's known in the    art distance statistic;-   3. Evaluation of the variation of the precision and accuracy values    calculated for the calibration curves for several potential linear    range definitions provided a visual evaluation of the linearity of    the relationship. There was no evidence of non-linearity of the    relationship inside of the chosen linear range 1-25 μg/ml.

Experiment 2E: Determination of Criteria for GA RS Standard Curve

Validation parameters derived from GA RS calibration curves, fittedwithin selected limits (1-25 μg/ml) of the linear range, were determinedto be the following:

-   1. R² of the linear regression fits of log₁₀ (IL-2 conc) to log₁₀    (GA conc) for each plate in the study;-   2. Slopes and intercepts for these straight line fits;-   3. Accuracy calculated at each calibration point for each plate; and-   4. Precision calculated at each calibration point for every plate.

In order to compute accuracy and precision, each calibration curve wasused to calibrate (back-calculate) the GA Concentrations given thevalues of IL-2 concentration:

X _(i-back)=10^((log) ₁₀ ^((IL-2 Conc)) _(i) ^(−α/β)

i=1, 2, 3—triplicate index.

The basic measure of (in)accuracy used was the percent differencebetween the mean of the estimates of concentration and the trueconcentration in the triplicate samples:

inaccuracy=([Mean(X _(i-back))−GA conc.]/GA conc.)*100%.

The basic measure of precision used was the relative standard deviation(RSD or CV) of the triplicate estimates of concentration:

precision=CV(X _(i-back))=[Std. Dev. (X _(i-back))/Mean(X_(i-back))]*100%.

The goal of the analysis was to propose acceptance criteria for thefitted calibration curve which ensured that the accuracy and precisionof the method were adequate. The acceptance criteria were based on theR² and the slope of the GA RS calibration curve. About 80% of the platescould be characterized by small inaccuracy values (<13%) and by goodprecision (1.1%-6.7%).

For these 16 “well behaved” standard curves, the following results wereobtained:

-   1. High R² values (>0.98);-   2. Relatively high slope values, reflecting dose response    relationships (>0.78 in 15 of 16 plates).

Since the majority of calibration curves were characterized byrelatively high R² (mean=0.99) and by relatively steep slopes(mean=0.87), in contrast to the excluded plates which had bothrelatively low R² (mean=0.94) and rather flat slopes (mean=0.72), theoverall acceptance criteria for calibration curves were considered interms of R² and slope. The simple rule defining the acceptanceparameters was based on the computation of cut-off points for the slopeand R² separately and located them mid-way between the maximum value forrejected curves (max_R²=0.95 max_slope=0.77) and the minimum value foraccepted curves (min_R²=0.98 min_slope=0.77). Thus, the acceptancecriteria were derived as follows:

-   1. R¹≧0.97;-   2. Slope≧0.77.

These criteria were applied to at least five different (triplicate)concentrations for fitting the calibration curve within the range 1-25μg/ml of GA concentration. Additionally, the range of intercept valueswas between 1.42-1.78, mean=1.58. This range was similar for the 16eligible and the 5 removed plates.

Accuracy and precision were calculated for each curve, and for eachconcentration among those on the plate. These individual values (foreach curve and concentration) were also averaged over:

-   1. Different concentrations for each curve;-   2. Different curves for each concentration; and-   3. Over all curves and concentrations.

The relevant conclusion was that for GA RS calibration curves based onat least five different calibration points in the linear range 1-25μg/ml, when the calibration curve was restricted to having R²≧0.97 andslope≧0.77, the resultant average accuracy and precision was estimatedas:

-   1. The mean (±SD) accuracy value for the method was: 8.0%±2.3%; and-   2. The mean (±SD) precision value for the method was: 2.9%±1.7%.

Reliability Assessment of GA RS Calibration Curves Based on DuplicateMeasurements

A comparison of the assay's accuracy and precision descriptivestatistics was performed in order to assess the reliability of GA RScalibration curves fitted using duplicate measurements at eachcalibration point. In addition, the individual accuracy and precisionvalues (for each curve and concentration) for all three possibleselections of duplicate measurements, out of the given triplicate, werestudied. When concentrating on those curves that satisfied theacceptance criteria described in the previous section and fitted withinthe limits of the defined linear range 1-25 μg/ml, it was evident thatwhen a triplicate measurement can not be provided for some reasons, itcan be successfully substituted by duplicate measurement.

TABLE 5 Accuracy and Precision Descriptive Statistics Summary TriplicateDuplicate (1, 2) Duplicate (1, 3) Duplicate (2, 3) Accuracy PrecisionAccuracy Precision Accuracy Precision Accuracy Precision Mean 8.00 2.938.13 2.53 7.98 2.88 8.40 2.40 S.D. 2.31 1.72 2.80 1.66 2.45 2.13 2.791.53 Min 5.23 1.10 4.93 0.95 5.21 0.93 4.97 0.81 Max 12.79 6.67 12.516.33 12.44 9.24 13.41 5.99

The mean (±SD) accuracy and precision of the method based on triplicateswere 8.0%±2.3% and 2.9%±1.7%, respectively (Table 5).

The mean (±SD) accuracy and precision of the method based on duplicateswere:

-   1. Accuracy: 8.1%±2.8%; Precision: 2.5%±1.7%;-   2. Accuracy: 8.0%±2.5%; Precision: 2.9%±2.1%; and-   3. Accuracy: 8.4%±2.8%; Precision: 2.4%±1.5%.

Experiment 2F: Determination of Statistical Relationship

It was found that the mean (in)accuracy of the method is 8.0% withSD=2.3%. The aim was to develop a reliable test for the slope comparisonof two log(dose)-log(response) lines of a new GA batch vs. GA RS. Thetest took into account the (in)accuracy of the above-mentioned method.The highest limit of the approximate 95% individual tolerance region forthe mean (in)accuracy of the method served as a threshold value:Mean+2*SD=12.6%. Thus, variations within the range±12.6% were considerednon-significant.

A full mathematical explanation of the relationship between β* (theslope of the batch line), β (the slope of the standard line) and thehighest permitted (in)accuracy value follows. Without loss ofgenerality, only the case where β*>β will be proved in detail (due tothe existing symmetry, the extension of the proof for the case whereβ*<β is obvious). The back-calculated dose value, for a givenlog(response) was:

X _(back)=10^((Y-α)/β) where Y=log₁₀ (IL-2 concentration).

The formula for the (in)accuracy calculation was:

(in)accuracy=[(10^((Y-α)/β) −X _(true))/X _(true)]*100%.

Y_(low) and Y_(high) were the lowest and highest log(response) valuespermitted by the highest allowable (in)accuracy of ±12.6%.

Thus, the region where the hypothesis of the equality of slopes was tobe accepted was:

$\quad\left\{ {\begin{matrix}{{\left\lbrack {\left( {10^{{({Y_{low} - \alpha})}/\beta}X_{1}} \right)/X_{1}} \right\rbrack*100\%} \geq {{- 12.6}\%}} \\{{\left\lbrack {\left( {10^{{({Y_{high} - \alpha})}/\beta}X_{2}} \right)/X_{2}} \right\rbrack*100\%} \leq {{- 12.6}\%}}\end{matrix}\left\{ \begin{matrix}{{10^{{({Y_{low} - \alpha})}/\beta}/X_{1}} \geq 0.874} \\{{10^{{({Y_{high} - \alpha})}/\beta}/X_{2}} \leq 1.126}\end{matrix} \right.} \right.$

Thus, the boundaries of the equality of the slopes were:

$\quad\left\{ \begin{matrix}{Y_{low} = {\alpha + {\beta \cdot {\log \left( {X_{1} \cdot 0.874} \right)}}}} \\{Y_{high} = {\alpha + {\beta \cdot {\log \left( {X_{2} \cdot 1.126} \right)}}}}\end{matrix} \right.$

The slope of a straight line was calculated as follows:

β*=(Y _(high) −Y _(low))/(log X ₂−log X ₁)=[β−log([X ₂ /X₁]·[1.126/08.74])]/log(X ₂ /X ₁)

β=[β·log(1.288)]/log(X ₂ /X ₁)=β·(1+log(1.288)/log(X ₂ /X ₁))

Assuming for the particular case under consideration that X₂/X₁=2 (fordose levels of 5 and 10 μg/ml), β* was calculated as follows:

β*=β·(1+log(1.288)/log 2)=β·1.365

Combining this result with the one obtained for the symmetric case whereβ*<β, the limits were calculated as:

$\quad\left\{ \begin{matrix}{\beta^{*} \leq {\beta \cdot 1.365}} \\{\beta^{*} \geq {\beta \cdot 0.635}}\end{matrix} \right.$

In the given data, all slope values were within the matching criticallimits, meaning that no deviation from the parallelism assumption wasobserved.

Once a batch was accepted as statistically valid (existence of linearityand parallelism has been proved), the potency ratio of the testpreparation relative to the standard was estimated. This was done in aparallel line assay by fitting straight parallel lines to the data anddetermining the horizontal distance between them:

${M = {{\log \; \rho} = {\frac{{\overset{\_}{Y}}_{T} - {\overset{\_}{Y}}_{S}}{B} - \left( {{\overset{\_}{X}}_{T} - {\overset{\_}{X}}_{S}} \right)}}};$

where ρdenoted the potency, Y_(S) , Y_(T) , X_(S) , X_(T) , were themean log(responses) and log(doses) of the standard and testpreparations, respectively. B—was a common slope for the standard andtest log(dose)−log(response) lines. The least-squares estimate of thecommon slope—B was a weighted average of the least-squares estimates oftwo slopes separately from the standard line and the test line. Takingthe anti-logarithm of the expression above, one was able to obtain apoint estimate of the “true % potency” of a test preparation relativelyto its standard:

${\% \mspace{14mu} {Potency}} = {{10\frac{{\overset{\_}{Y}}_{T} - {\overset{\_}{Y}}_{S}}{\beta}} - {{\left( {{\overset{\_}{X}}_{T} - {\overset{\_}{X}}_{S}} \right) \cdot 100}{\%.}}}$

Example 3 Validation of the Standard Procedure of Example 1

The goal of the analysis, presented below, was to establish validatedrelease specifications for the relative potency of a GA batch. A GAbatch was considered valid, if the following criteria, based onstatistical inference, were fulfilled:

-   1. No violations of the assumptions involved in the bioassay    analysis approach:    -   (a) Independence and normality of the log(responses);    -   (b) Homogeneity of the variance of the log(responses);    -   (c) No outliers;    -   (d) Parallelism (non-significance of the slope ratio test);-   2. The point estimate of the relative potency was within a    pre-specified range: 80%-125%; and-   3. The 95% Fiducial Limits for the “true relative potency” value    were within a wider pre-defined confidence range: 70% to 143%.

The model assumed that the standard and the test preparations shouldbehave as if one were a simple dilution of the other. This means thatthe log(dose)-response lines for the two preparations should not deviatesignificantly from linearity and parallelism. Thus, an anti-logarithm ofthe constant horizontal displacement between these straight lines wasable to serve as an estimate of the potency ratio. These tworequirements, linearity and parallelism, constituted a concept of theassay validity. The check of validity was a prerequiste to theestimation of the relative potency and its fiducial limits.

The estimate of random error was needed for the computation of fiduciallimits for the true value of the relative potency. This measure wasobtained by the implementation of the statistical technique known as“Analysis of Variance” (ANOVA). Therefore, the classical statisticalassumptions of the ANOVA must have been satisfied. The requirements forthe statistical analysis of a parallel-line bioassay model were asfollows:

-   1. The responses were independently normally distributed about their    expected values;-   2. The variance of the response was not affected by the mean    response value;-   3. There were no outliers;-   4. The relationship between the log(dose) and response was able to    represented by a straight line over the range of doses; and-   5. The straight line of the test preparation was parallel to that of    the standard.

The batch analysis data was obtained from different experimentsperformed on different days by different operators. Validation trials ofthe standard procedure of Example 1 were carried out by a series ofexperiments, each involving: 1) immunization of mice with 250 μg GA RSin CFA; 2) preparation of a primary culture from the LN cells 9-11 daysfollowing immunization; 3) incubation of the LN cells with variousconcentrations of GA RS and with test samples; 4) collection of theculture media and analysis of IL-2 levels by ELISA; 5) plotting a GA RScurve based on triplicate IL-2 measurements performed at 6 dose levelsfrom 1-25 μg/ml; and 6) comparison of the T cell response to each testsample to the response to the RS batch (in triplicate) at twoconcentrations within the linear range (5 and 10 μg/ml). The % CV wasalso calculated for each triplicate in order to detect any problemsassociated with variability between triplicates (normally, the % CVbetween triplicates should not exceed 10-15%). For the given data, noviolations of the conditions were detected.

The validation characteristics used to provide an overall knowledge ofthe capabilities of the analytical procedure were: linearity, range,accuracy, precision, specificity and robustness. The validation criteriaand analyses were based on the ICH consensus guideline, “Validation ofAnalytical Procedures: Methodology”, November 1996 (CPMP/ICH/281/95).

Statistical methods recommended in “European Pharmacopoeia” guidelinewere adapted to the given data for analysis purposes.

Experiment 3A: Linearity and Range

In each in vitro test, a dose-response curve of GA RS batch was used tocalculate the relative response of the cells to the tested samples. Eachcalibration curve included at least five points (without zero).Twenty-one calibration curves collected from different in vitro tests,performed during the development and the validation stages, were plottedand evaluated for each plate.

Statistical analysis of the data revealed that the plots of log₁₀ (IL-2concentration) versus log₁₀ (GA RS concentration) provided the bestlinear fit. The linear range mainly emerged by visual inspection andevaluation of accuracy and precision of the calibration points. The %RSD was calculated for each triplicate in order to detect any problemsassociated with variability between triplicates (normally, the % RSDbetween triplicates should not exceed 10-15%). The range of GA RS curvewas specified between 1-25 μg/ml.

Based on these analyses, the GA RS curve should be comprised of at least6 calibration points, one with zero concentration (negative control) andat least 5 concentrations of GA RS in the range between 1 and 25 μg/ml.Linear regression of log₁₀ (IL-2 concentration) versus log₁₀ (GA RSconcentration) should have an R²≧0.97 and a slope≧0.77.

Experiment 3B: Accuracy

The accuracy of the method was established across the specified linearrange of the GA RS curve. Statistical analysis of the data revealed thatthe mean accuracy of the method was: 8.0%±2.3%.

Experiment 3C: Precision

The basic measure of precision used was the relative standard deviation(RSD) of replicate (usually triplicate) estimate of concentration.

The RSD was established across the linear range of GA RS curves.Statistical analyses of the data revealed that the mean precision of themethod was 2.9%±1.7%. The reliability of duplicate measures wasequivalent to that of triplicate. Therefore, when one of the threereplicates was identified as an outlier, the outlier was omitted and theresults from duplicate measures were accepted.

Experiment 3D: Method Repeatability

The GA specific T cell response to a GA DS batch was measuredrepeatedly, 3 times, in the same in vitro test. Three weights of thesame batch were each diluted to 5 and 10 μg/ml and incubated with theGA-specific T-cells. The levels of IL-2 in the culture media of the testsamples and of the GA RS samples, were measured by ELISA in triplicate.The % potency and 95% fiducial limits of the cells to each replicatewere calculated relative to the GA RS. Table 6 shows the % responsecalculated for each replicate.

TABLE 6 Method Repeatability 95% Fiducial GA DS Limits (Lower Sampleconc. AVG Limit-Upper # (μg/ml) % Potency N = 6 SD RSD Limit) 1 5 75 8074 77 3.3 4.2 67-89 10 73 79 81 2 5 83 83 92 84 5.0 5.9 73-97 10 80 7988 3 5 72 74 71 76 5.2 5.2 65-88 10 80 78 79

Experiment 3E: Intermediate Precision

The % response of a GA DS batch was tested in 3 different in vitroteats, performed in different days, by 3 different investigators fromthe same laboratory. Table 7 summarizes the % potency and 95% fiduciallimits determined for this batch in the 3 repeated experiments.

TABLE 7 Intermediate Precision 95% Fiducial GA DS Limits (Lower Testconc. AVG Limit-Upper # (μg/ml) % Potency N = 6 SD RSD Limit) 1 5 85 8683 83 3.1 3.7 71-97 10 77 84 84 2 5 90 86 87 86 3.0 3.7 79-94 10 82 8489 3 5 75 80 74 77 3.3 4.2 65-88 10 73 79 81

Experiment 3F: Method Reproducibility

The reproducibility of the method was assessed by means ofinter-laboratory study. The % response GA DS batch was tested in twodifferent experiments, performed in 2 different laboratories, usingdifferent analysts, equipment and reagents. Table 8 summarizes theresults from both labs.

TABLE 8 Method Reproducibility Lab 1 Lab 2 Avg % Potency ± RSD 86 ± 3.583 ± 5.0 %95 Fiducial Limits 79-94 78-90

Based on the above experiments, it can be concluded that the in vitrotest is reproducible.

Experiment 3G: Specificity

The discrimination of the method was tested at 3 levels: 1)discrimination between samples incubated with/without GA RS (matrixeffect); 2) discrimination between GA RS and other related andnon-related proteins and peptides, including GA DS; and 3)discrimination between GA RS and GA related copolymers in which thepeptide sequences have been deliberately modified.

i) Recognition of GA RS by GA RS-Specific T Cells (Matrix Effect)

GA-specific T cells were induced by immunization of female(SJL×BALB/C)F1 mice mouse with 250 g GA RS in CFA. This dose of GA isroutinely used for testing the biological activity of GA batches in thismouse strain using the EAE blocking test. The control group in thisexperiment was injected with CFA alone. Ten days following immunization,LN cells were removed from the both groups of mice. The cells wereincubated with GA RS for 18-24 hours at 37° C. in a 5% CO₂ humidifiedincubator.

Subsequently, the cultures were centrifuged and the supernatantscollected and assayed for interleukin-2 (IL-2, a cytokine secreted fromactivated T cells) by ELISA using biotinylated antibodies specific toIL-2 and strepavidin-horseradish peroxidase (HRP) conjugate fordetection (FIG. 1). Each plate run included blank control (first andsecond antibodies without the cytokine standard). Each plate run alsoincluded quality control (QC) samples (three concentrations of cytokinestandard within the assay's linear range). Each in vitro test included apositive control (Con A, a non-specific T-cell stimulant) and a negativecontrol (no GA or any other antigen). FIG. 2 shows that the LN cellsfrom mice immunized with GA RS secrete IL-2 dose dependently in responseto GA RS in culture, while LN cells from the control mice do not respondto GA. RS in culture. The levels of IL-2 in the negative control samplesis usually below or close to the ELISA detection limit (approximately 3μg/ml). These IL-2 levels are always below the levels secreted by thelowest calibration point of GA RS (1 μg/ml). These results indicate thatthe secretion of IL-2 by the GA specific T-cells is GA dependent.

ii) Discrimination between related and non-related antigens

The discrimination between related and non-related antigens (proteinsand single peptides) was demonstrated by testing the response of the GARS-specific T cells to various antigens in-vitro. A primary culture ofLN cells derived from female (SJL×BALB/C)F1 mice immunized 9-11 daysearlier with 250 μg GA RS in CFA. The primary culture was incubatedovernight with GA RS and with various other antigens at 37° C. in a 5%CO₂ humidified incubator. Then, the cultures were centrifuged and thesupernatants collected and assayed for IL-2 by ELISA as in Experiment3G(i).

Table 9 shows that in this experimental system the GA-specific T cellsdid not respond to either human MBP (myelin basic protein), the MBPimmunodominant peptide pp. 87-99 (an encephalitogenic peptide), or itsanalog pp. 87-99_(Ala 76) (an EAE suppressor peptide). Lysozyme, anon-relevant basic protein, was also not recognized by the GA-specific Tcells. TV-35 and TV-109 were peptides with a molecular weight of 3757and 11727, respectively (PCT International Publication No. WO 00/18794).These peptides had a defined sequence comprised from the same four aminoacids of GA (Ala, Glu, Lys, Tyr), in the same molar ratio as in GA. TheGA RS-specific LN cells did not respond to TV-35, and had a very lowcross-reactivity with TV-109. These results can be explained by theobservation that immunization with GA RS induced the formation of amixture of T cells with different specificity towards the multipleT-cell epitopes present in GA. TV-35 and TV-109 may share commonsequences with GA, however, and incubation of the GA-specific T cellswith a single peptide probably caused only a partial stimulation of asmall fraction of the GA-specific cells in culture. Thus, the overallT-cell response (secretion of IL-2) was below or close to detectionlimits.

TABLE 9 Specificity of GA RS-specific LN cells Antigen % Potency¹ GA RS100 Lysozyme 0 Human MBP 0 MBP pp. 87-99 0 MBP pp. 87-99_(Ala 96) 0TV-35 0  TV-109 17${{\,^{1}\%}\mspace{14mu} {Potency}} = \frac{{IL}\text{-}2\mspace{14mu} {concentration}\mspace{14mu} {in}\mspace{14mu} {culture}\mspace{14mu} {media}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {antigen} \times 100}{{IL}\text{-}2\mspace{14mu} {concentration}\mspace{14mu} {in}\mspace{14mu} {culture}\mspace{14mu} {media}\mspace{14mu} {of}\mspace{14mu} {GA}\mspace{14mu} {RS}}$

The in vitro test was sensitive to the average molecular weight (MW) ofthe GA batch. FIG. 11 shows the response of the GA RS-specific cells toGA RS (MW=7900) and to GA DS batches differing in their average MW. Ascan be seen, the response generally correlated with the average MW; thehigher the average MW, the greater the response. However, it should benoted that the release specifications for the average MW of GA DS arebetween 4700-10000, and that similar levels of IL-2 were secreted inresponse to DS batches with average MW within specifications (FIG. 11).These results indicate that the method was highly specific to GA andsensitive to changes in the average MW of GA.

iii) Recognition of GA drug substance (DS) and Copaxone® Drug Product(DP) by GA RS-Specific T Cells

Nine to eleven days following immunization of female (SJL×BALB/C)F1 micewith 250 μg GA RS in CFA, the LN cells were removed and cultured withvarious doses of GA RS batch (the immunizing antigen) and with a DSbatch.

IL-2 was measured as in Experiment 3G(i). FIG. 12A shows that the LNcells cross-reacted with both standard batches. The dose-response curvesof IL-2 secretion (measured by ELISA as above) by both batches weresimilar, indicating that the tested batches shared similar T-cellepitopes. Comparison between GA RS and a Copaxone® batch shows that theGA RS-specific T cells also cross-reacted with the DP batch, and thatmannitol, the excipient in the Copaxone® formulation, did not affect orinterfere with the T-cell responses FIG. 12B). Thus, this methodprovides an indication of batch-to-batch reproducibility.

v) Discrimination Between GA and Related Copolymers

In Experiment 3G(ii), it was demonstrated that the in vitro test was,sensitive to the average MW of GA peptides, using GA DS batchesdiffering in their average MW. Since the experiment was based onbio-recognition of GA by GA-specific T cells, which specifically respondto linear sequences, it was expected that the method would be sensitiveto variations/modifications in the sequences of GA peptides. This wasdemonstrated by using: 1) copolymers synthesized from only 3 out of the4 amino acids comprising GA; 2) a GA batch (XX) resulting fromdeliberate modification in manufacturing conditions, i.e., addition ofexcess of free amino acids to GA monomers during synthesis. The averageMW of this batch was high and out of specifications (MW=11150 Da); and3) degradation products of GA RS obtained by proteolysis with trypsinand chymotrypsin.

Table 10 shows that the GA-specific T-cells did not respond to the 3amino acid copolymers lacking lysine, alanine or tyrosine. In addition,the % response of the cells to the batch XX was relatively high and outof the method specifications (100±30%), indicating that the method mightbe sensitive to modifications in the production process. The high %response can also be explained by the sensitivity of the test to the MWof GA peptides, as demonstrated.

TABLE 10 Method Specificity Copolymer Modification Average % Potency ±RSD Tyr-Lys-Glu Lacking Alanine 0 Tyr-Ala-Glu Lacking Lysine 0Ala-Glu-Lys Lacking Tyrosine 0 Batch XX Excess of free amino 170 ± 4.7acids in polymerization stage

Kinetics studies of GA RS proteolysis by trypsin and chymotrypsin showthat the in vitro test was sensitive to degradation of GA peptides.FIGS. 13 and 15 show that the secretion of IL-2 by the cells was reducedupon proteolysis time, and the % potency of the cells to the proteolysedpeptides was out of the method specifications (100±30%) (Table 11).Overlay chromatograms (by RP-HPLC) of the degraded samples (FIGS. 14 and16) demonstrated the kinetics of the proteolysis by trypsin andchymotrypsin, respectively. The cumulative results from all specificitystudies revealed that the method was highly specific to GA anddiscriminated between GA and closely related antigens.

TABLE 11 Method Specificity - Effect of Proteolysis Time of proteolysisEnzyme (minutes) Average % Potency Trypsin 1 40 5 36 15 19 30 7overnight 0 Chymotrypsin 5 11 20 0

Experiment 3G: Robustness i) Robustness of Acceptance Criteria

The consistency and robustness of the defined acceptance criteria wasexamined by comparing the resulting estimates of the relative potencyobtained for the repeated GA batches. The batch analysis data included anumber of repeated GA batches. Two batches were measured on threedifferent days by different operators. One batch was tested on twodifferent days by different operators, as well.

In the parallelism test for the repeated GA batches, all GA batch slopesvalues were within the appropriate critical limits for the parallelismslope ratio test. All GA batches satisfied the acceptance criteria forthe point estimates of the relative potency values with 95% fiduciallimits (the estimated % potency was within the limits of 80%-125% andthe 95% fiducial limits were within the range of 70%-143%). For theanalyzed data of the repeated GA batches, their validity did not dependon the day of experiment or the operator performing the test. This datasupports the robustness of established specifications.

ii) Robustness of Critical Parameters in the Immunization Procedure andthe In Vitro Reaction

The robustness of critical parameters in both the immunization procedureand the in vitro reaction was evaluated. Briefly, it was shown that: 1)the immunological response of the LN cells was not affected by theimmunizing dose of GA RS; 2) the immunization period was 9-11 days; 3)the response of the LN cells to GA RS was higher compared to the spleencells response; 4) immunization with GA RS+CFA resulted in the LN cellshaving a stronger response compared to immunization with ICFA; 5) thepresence of serum in culture media strongly affected the GA-specific Tcell response, thus the in vitro reaction was performed in a serum-freemedia; 6) the optimal time frame for collecting the culture media was18-21 hours following incubation with GA RS and test samples; and 7) theculture media can be kept at −20° C. for up to one week before tested inELISA. Thus, it was shown that the method was robust.

Summary Statistics for the Point Estimate and 95% Fiducial Limits ofRelative Potency 95% Tolerance Limits for the Mean Relative Potency

To assess the acceptance limits for the estimated relative potency of anew batch, the mean and the standard deviation of the individuallog(potency) estimates were calculated:

Mean(M _(i))=0.0074;

SD(M _(i))=0.0402.

An approximated 95% tolerance range for the mean relative potency value,based on the analyzed data, was:

[10^(Mean(M) ^(i) ^(±2*SD(M) ^(i) ⁾]*100%=[84%,122%].

Range of the 95% Fiducial Limits of Relative Potency

The minimum and maximum values of the 95% Fiducial Limits for theindividual relative potency estimates were:

-   -   Minimum(Low Limit)=79.3%    -   Maximum(High Limit)=147.3%

Satisfaction of the Acceptance Criteria

Based on the analysis, the acceptance criteria were determined to be:

-   -   1. The assumptions involved in bioassay analysis approach were        fulfilled, namely:        -   a. Independence and normality of the log(responses);        -   b. Homogeneity of the variance of the log(responses);        -   c. No outliers; and        -   d. Parallelism (non-significance of the slope ratio test).    -   2. The estimated relative potency was not less than 80% and not        more than 125% of the standard potency; and    -   3. The 95% Fiducial Limits of error of the estimated relative        potency were not less than 70% and not more than 143% of the        standard potency.

Discussion of Example 3

Validation of the in vitro test revealed that the method wasreproducible and the mean accuracy and precision were in an acceptablerange. The method was highly specific to GA peptides and sensitive tothe quality of the active substance.

Summary and Discussion

An in vitro method was developed for GA DS and Copaxone® batches. Thismethod was based on bio-recognition of T-cell epitopes (linearsequences) by GA RS-specific T cells. The GA RS-specific T cells secreteTh₀ cytokines in response to GA in culture. In this method, therecognition of GA batches by T cells is monitored by measuring thelevels of IL-2 in the culture media by ELISA. It was shown that the GARS-specific T cells are cross-reactive with both DS and DP batches,indicating that these batches share similar sequences with the RS batch,and that mannitol, the excipient in the DP formulation, does notinterfere with the reaction.

The method was very specific to GA peptides and is sensitive to theaverage MW of the peptide mixture. MBP was not recognized by theGA-specific T cells. MBP immunodominant peptides (both encephalitogenicand suppressive peptides), as well as single peptides with amino-acidcomposition similar to that of GA, did not stimulate the T cells.Critical parameters in the immunization procedure, as well as in thein-vitro reaction, were optimized during this experiment. Thisexperiment showed that the method was very reproducible and robust.

The method can be adapted to standardize other T cell antigens for usein pharmaceutical compositions. A primary culture of T cells specific toan antigen RS, instead of GA RS, can be made from animals immunizedagainst the antigen RS. The cytokine production of this culture inresponse to antigen RS and in response to the sample antigen can bemeasured. The cytokine production in response to antigen RS can beplotted against the concentration of antigen RS to create a standardcurve. The cytokine production in response to the sample antigen can becompared to the standard curve to determine whether the antigen iswithin the acceptable range of potency.

The optimum cytokine to monitor can be determined as in Experiment 2A.Conditions for immunization and the in vitro test may be optimized as inExperiments 2B and C.

REFERENCES

-   U.S. Pat. No. 3,849,550, issued Nov. 19, 1974 (Teitelbaum, et al.).-   U.S. Pat. No. 5,800,808, issued Sep. 1, 1998 (Konfino, et al.).-   PCT International Publication No. WO 00/05250, published Feb. 3,    2000 (Aharoni et al.).-   PCT International Publication No. WO 00/18794-   Aharoni, R. et al., T suppressor hybridomas and    interleukin-2-dependent lines induced by copolymer 1 or by spinal    cord homogenate down-regulate experimental allergic    encephalomyelitis, Eur. J. Immunol., (1993), 23: 17-25.-   Bornstein, et al., New Eng. J. Med., 1987, 317(7), 408-414.-   Johnson, K. P., Neurology, 1:65-70 (1995).-   Lando et al., Effect of cyclophosphamide on suppressor cell activity    in mice unresponsive to EAE, J. Immunol. (1979) 123(5): 2156-2160.”-   Lisak et al., In vitro and in-vivo immune responses to homologous    myelin basic protein in EAE, Cell. Immunol. (1974) 11:212-220.    “Copaxone” in Physician's Desk Reference, 2000, Medical Economics    Co., Inc., Montvale, N.J., 3115.-   “Validation of Analytical Procedures: Methodology”, November 1996    (CPMP/ICH/281/95).-   European Pharmacopoeia, 1997.

1. A process for measuring the potency of a test batch of glatirameracetate relative to the known potency of a reference batch whichcomprises a. immunizing female (SJLXBALB/C)F1 mice between 8 and 12weeks of age with a predetermined amount of glatiramer acetate from thereference batch. b. preparing a primary culture of lymph node cells fromthe mice of step (a) 9-11 days after immunization; c. separatelyincubating at least five reference samples, each of which contains apredetermined number of cells from the primary culture of step (b) and apredetermined amount of glatiramer acetate between 1 μg/ml and 25 μg/mlfrom a reference batch; d. incubating at least two samples, each ofwhich contains a predetermined number of cells from the primary cultureof step (b) and a predetermined amount of glatiramer acetate from thetest batch; e. determining for each sample in steps (c) and (d), theamount of interleukin-2 secreted by the cells in each sample after 18-21hours of incubation of such sample; f. correlating the amounts ofinterleukin-2 secreted by the samples incubated with the test batch ofglatiramer acetate with the amounts of interleukin-2 secreted by thesamples incubated with the reference batch of glatiramer acetate so asto determine the potency of the test batch of glatiramer acetaterelative to the reference batch of glatiramer acetate, wherein in eachsample in steps (c) and (d), the predetermined number of cells issubstantially identical, and wherein for each sample containing apredetermined amount of glatiramer acetate from the test batch there isa corresponding reference sample containing a substantially identicalpredetermined amount of glatiramer acetate from the reference batch. 2.The process of claim 1, wherein six reference samples are separatelyincubated in step (d).
 3. A process for measuring the potency of a testbatch of glatiramer acetate relative to the known potency of a referencebatch which comprises a. immunizing a test mammal with a predeterminedamount of glatiramer acetate from the reference batch; b. preparing aprimary culture of cells from the test mammal of step (a) at apredetermined time after immunization; c. separately incubating at leasttwo reference samples, each of which contains a predetermined number ofcells from the primary culture of step (b) and a predetermined amount ofglatiramer acetate from a reference batch; d. incubating at least twosamples, each of which contains a predetermined number of cells from theprimary culture of step (b) and a predetermined amount of glatirameracetate from the test batch; e. determining for each sample in steps (c)and (d), the amount of a cytokine secreted by the cells in each sampleafter a predetermined time period of incubation of such sample; f.correlating the amounts of the cytokine secreted by the samplesincubated with the test batch of glatiramer acetate with the amounts ofthe cytokine secreted by the samples incubated with the reference batchof glatiramer acetate so as to determine the potency of the test batchof glatiramer acetate relative to the reference batch of glatirameracetate, wherein in each sample in steps (c) and (d), the predeterminednumber of cells is substantially identical, and wherein for eachimmunization sample containing a predetermined amount of glatirameracetate from the test batch there is a corresponding reference samplecontaining a substantially identical predetermined amount of glatirameracetate from the reference batch.
 4. The process of claim 3, wherein thecytokine is an interleukin.
 5. The process of claim 4, wherein theinterleukin is interleukin-2.
 6. The process of claim 4, wherein theinterleukin is interleukin-6.
 7. The process of claim 4, wherein theinterleukin is interleukin-10.
 8. The process of claim 3, wherein thecytokine is interferon-gamma.
 9. The process of claim 3, wherein themammal produces T cells specific to glatiramer acetate referencestandard.
 10. The process of claim 3, wherein the mammal is a rodent.11. The process of claim 10, wherein the rodent is a mouse.
 12. Theprocess of claim 11, wherein the mouse is a female (SJLXBALB/C)F1 mouse.13. The process of claim 3, wherein the mammal is about 8 to about 12weeks old.
 14. The process of claim 3, wherein the cells are lymph nodecells.
 15. The process of claim 3, wherein the cells are spleen cells.16. A process for preparing a batch of glatiramer acetate as acceptablefor pharmaceutical use which comprises a. preparing a batch ofglatiramer acetate; b. measuring the relative potency of the batchaccording to the process of claim 1; and c. qualifying the batch asacceptable for pharmaceutical use if the relative potency so measured isbetween 80% and 125% of the reference batch of glatiramer acetate.
 17. Aprocess for preparing glatiramer acetate acceptable for pharmaceuticaluse which comprises a. preparing a batch of glatiramer acetate; b.measuring the relative potency of the batch according to the process ofclaim 3; and c. qualifying the batch as acceptable for pharmaceuticaluse if the relative potency so measured is between 80% and 125% of thereference batch of glatiramer acetate.